Council Directive 2009/156/ECShow full title

[F1 2. Real-time RT-PCR Procedure U.K.

2.1. Group-specific real-time RT-PCR by Agüero et al., 2008 (1) U.K.

This group-specific real-time RT-PCR targets VP7 of the AHSV and is able to detect all known AHSV serotypes and strains currently circulating. It has been employed with very good results by the participating national reference laboratories of the European Union Member States in the proficiency tests annually organised by the European Union Reference Laboratory for the period 2009-2015. Moreover, in an international ring trial organised in 2015 in the framework of the OIE reference laboratories network this protocol was ranked very high amongst others.

Primer and probe sequences for the detection of AHSV species viruses:

— forward Primer

5′-CCA-GTA-GGC-CAG-ATC-AAC-AG-3′

— reverse Primer

5′-CTA-ATG-AAA-GCG-GTG-ACC-GT-3′

— MGB-TaqMan probe

5′-FAM-GCT-AGC-AGC-CTA-CCA-CTA-MGB-3′

2.1.1.

Primer stock concentration is diluted to a working concentration of 8 μM ( ‘ primer working stock 8 μM ’ ) whereas probe is diluted to a working concentration of 50 μM ( ‘ probe working stock 50 μM ’ ). A test plate layout should be designed and loaded into the real time PCR machine software. Using the layout as a guide, 2,5 μl of each primer working stock 8 μM is added to each well that will contain RNA samples, positive and/or negative controls (final concentration of the primer will be 1 μM in the 20 μl RT-PCR mix). The plate is held on ice.

2.1.2.

2 μl of isolated RNA (test samples and positive control), or 2 μl of RNAse-free water in negative reaction controls, is mixed with forward and reverse primers. This mixture is denatured by heating at 95 °C for 5 minutes, followed by rapid cooling on ice for at least 5 minutes.

2.1.3.

An appropriate volume of real time one-step RT-PCR master mix for the number of samples to be tested is prepared following manufacturer's instructions. 0,1 μl of probe working stock 50 μM is added to each well containing RNA samples (final concentration of the probe will be 0,25 μM in each well containing RNA samples). 13 μl of real time one-step RT-PCR master mix is distributed in each well on the PCR plate containing the denatured primers and RNA.

2.1.4.

The plate is placed in a real time thermal cycler programmed for reverse transcription and cDNA amplification/fluorescence detection. Amplification conditions consist of a first reverse-transcription step at 48 °C for 25 minutes, followed by 10 minutes at 95 °C ( ‘ hot start ’ ) and 40 cycles of 15 seconds at 95 °C, 35 seconds at 55 °C and 30 seconds at 72 °C (or 40 cycles at 97 °C for 2 seconds and 55 °C for 30 seconds if reagents and thermocycler allowing fast reactions are used). Fluorescence data are acquired at the end of the 55 °C step.

2.1.5.

The assay is considered not valid if atypical amplification curves are obtained, and must be repeated.

Samples are considered positives, if the Ct value (cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold) is lower than or equal to the defined Ct threshold (35) within 40 PCR cycles (Ct ≤ 35).

Samples are considered inconclusive, if the Ct value is higher than the defined Ct threshold (35) within 40 PCR cycles (Ct > 35).

Samples are considered negative, if a horizontal amplification curve is obtained which does not cross the threshold line within 40 PCR cycles.

2.2. Group-specific real-time RT-PCR by Guthrie et al., 2013 (2) U.K.

Real-time RT-PCR using fluorescence resonance energy transfer (FRET) probes to detect nucleic acid of AHSV.

The AHSV RT-PCR assay described was designed using sequences from a wide variety of currently circulating field strains of AHSV (Quan et al., 2010 (3) ). It also incorporates a proprietary synthetic external control assay to verify proper functioning of the assay components.

Kits for the one-step real-time PCR are available commercially. Below are some basic steps as described by Guthrie et al. (2013), which can be modified depending upon local/case-specific requirements, kits used and equipment available.

Primer and probe sequences for the detection of AHSV species viruses:

— forward Primer

5′-AGA-GCT-CTT-GTG-CTA-GCA-GCC-T-3′

— reverse Primer

5′-GAA-CCG-ACG-CGA-CAC-TAA-TGA-3′

— MGB-TaqMan probe

5′-FAM-TGC-ACG-GTC-ACC-GCT-MGB-3′

2.2.1.

Primer and probe mix stock solutions are made up in a 25× concentration at 5 μΜ for the forward and reverse primers and 3 μΜ for the probe. A test plate layout should be designed and loaded into the real-time PCR machine software. Using the layout as a guide, 5 μl of RNA samples, including test samples and positive and negative controls, are added to appropriate wells of the plate following the layout.

2.2.2.

The RNA is denatured by heating at 95 °C for 5 minutes, followed by rapid cooling on ice for at least 3 minutes.

2.2.3.

An appropriate volume of real-time one-step RT-PCR master mix for the number of samples to be tested is prepared, following the manufacturer's instructions. 1 μl of 25× primer probe mix stock solution (from point 2.2.1 above) is included in the master mix to give a final concentration in each well of 200 nM for each primer and 120 nM of the probe. 20 μl of the master mix is distributed in each well on the PCR plate containing the denatured RNA.

2.2.4.

The plate is placed in a real-time thermal cycler programmed for reverse transcription and cDNA amplification/fluorescence detection as suggested by the manufacturers. Amplification conditions consist of, for example, a first reverse-transcription step at 48 °C for 10 minutes, followed by 10 minutes at 95 °C and 40 cycles of 15 seconds at 95 °C and 45 seconds at 60 °C.

2.2.5.

Samples are considered positives, if the normalised fluorescence for the AHSV RT-PCR assay exceeds a 0,1 threshold within 36 PCR cycles in all replicates of a sample.

Samples are considered inconclusive, if the normalised fluorescence for the AHSV RT-PCR assay exceeds a 0,1 threshold between 36 and 40 PCR cycles in any replicate of a sample.

Samples are considered negative, if the normalised fluorescence for the AHSV RT-PCR assay did not exceed a 0,1 threshold within 40 PCR cycles in all replicates of a sample and if the normalised fluorescence for the proprietary synthetic external control assay exceeded a 0,1 threshold within 33 PCR cycles.]

Textual Amendments

F1 Substituted by Commission Implementing Decision (EU) 2016/1840 of 14 October 2016 amending Annex IV to Council Directive 2009/156/EC as regards methods for African horse sickness diagnosis (notified under document C(2016) 6509) (Text with EEA relevance).

(1)

[F1 Agüero M., Gomez-Tejedor C., Angeles Cubillo M., Rubio C., Romero E. and Jimenez-Clavero A. (2008). Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. J. Vet. Diagn. Invest., 20, 325-328.]

(2)

[F1 Guthrie AJ, MacLachlan NJ, Joone C, Lourens CW, Weyer CT, Quan M, Monyai MS, Gardner IA. Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus. Journal of Virological Methods. 2013;189(1):30-5.]

(3)

[F1 Quan, M., Lourens, C.W., MacLachlan, N.J., Gardner, I.A., Guthrie, A.J., 2010. Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus. J. Virol. Methods 167, 45-52.]

Textual Amendments

F1 Substituted by Commission Implementing Decision (EU) 2016/1840 of 14 October 2016 amending Annex IV to Council Directive 2009/156/EC as regards methods for African horse sickness diagnosis (notified under document C(2016) 6509) (Text with EEA relevance).